Research this past year focused on the molecular characterization of glutamate receptors and their function in the auditory system. Based on pharmacological properties and sequence similarities, it is now known that there are three families of ionotropic glutamate receptors: AMPA receptors comprised of subunits GluR1-4; kainate receptors comprised of subunits GluR5-7 and KA 1&2; and NMDA receptors comprised of subunits of NMDAR1 and NMDAR1A-D. Our efforts to develop subunit-specific antibodies to these proteins in order to study their distributions and biochemical properties continued as antibodies selective for NMDAR1, GluR6 and KA2 were made. Using these antibodies it was shown that GluR6 and KA2 form an immunoprecipitable molecular complex in brain and transfected cells. Our studies also showed that to a limited extent complexes can also form between members of the AMPA and kainate families. Applying these antibodies to immunocytochemical studies, we found that GluR6, KA2 and NNMDAR1 were widely distributed throughout the brain. At the ultrastructural level, all antibodies stained postsynaptic densities in cerebral cortex, hippocampus and cerebellum. A variant of GluR3, sGluR3, detected in cochlear nRNA, was characterized and found to be missing a 33 amino acid segment in the second cytoplasmic loop. In the oocyte expression system, sGluR3 had very little ion channel activity when expressed alone. When expressed with other AMPA receptor subunits, sGluR3 diminished their activities suggesting that it assembles with normal subunits to produce a nonfunctional receptor complex. In situ hybridization histochemistry, PCR amplification and immunocytochemistry are used to study glutamate receptors expressed in the cochlea and auditory nuclei of the brain stem. In the cochlea our results suggest that AMPA, kainate and NMDA receptors are expressed in spiral ganglion neurons. Since most neurons appear to express members of each receptor family, these results imply that all three receptor types are involved in synaptic transmission at the hair cell/auditory nerve synapse. In the cochlear nucleus, our efforts are continuing to identify the receptor subunits expressed and the neurons in which they are expressed. In situ hybridization showed that essentially all glutamate receptor subunits are expressed in the cochlear nucleus, but expression varies considerably among neuron types. AMPA, kainate and NMDA receptors appear to have distinct, but overlapping patters of expression.